This invention relates to gene therapy. More particularly, the invention relates to a composition of polyethylene glycol-grafted poly-L-lysine and a hepatocyte cell targeting molecule-polyethylene glycol-grafted poly-L-lysine for gene delivery to a hepatocyte cell.
Twenty-five years ago Friedmann outlined prospects for human gene therapy. (T. Friedmann and R. Roblin (1972) Gene Therapy for Human Genetic Disease? 175 Science 949-955 (1972). Since then, gene therapy has represented a new paradigm for therapy of human disease and for drug delivery. The implicit emphasis of prior research has been on determining the safety of gene transfer procedures, often placing efficacy as a secondary goal. A major technical impediment to the gene transfer is the lack of an ideal gene delivery system. If it were possible to deliver the gene to the appropriate specific cells in sufficient quantities without adverse side effects, gene therapy would be efficacious. Currently very few organs or cells can be specifically targeted for gene delivery. There are many established protocols for transferring genes into cells, including calcium phosphate precipitation, electroporation, particle bombardment, liposomal delivery, viral-vector delivery, and receptor-mediated gene delivery. MS Wadhwa, Targeted Gene Delivery with a Low Molecular Weight Glycopeptide Carrier. 6 Bioconj. Chem. 283-291 (1995). Although all of these methods can be used for mammalian cultured cells, there are many difficulties in introducing genes into target cells in vivo.
Transfection methods using retroviral or adenoviral vectors overcome some of these limitations. Retroviral vectors, in particular, have been used successfully for introducing exogenous genes into the genomes of actively dividing cells such that stable transformants are obtained. D. G. Miller et al., Gene Transfer by Retrovirus Vectors Occurs Only in Cells that are Actively Replicating at the Time of Infection. 10 Mol. Cell Biol. 4239-4242 (1990). Viral vector systems often involve complementation of defective vectors by genes inserted into `helper` cell lines to generate the transducing infectious agent. However, it is well known that the host immune response to adenoviruses limits their use as a transfer facilitating agent to a single administration. To address this limitation, fusion peptides of the influenza virus hemagglutinin have been employed to replace adenoviruses as endosomal lytic agents, but with limited success. S. Gottschalk et al. A Novel DNA-Peptide Complex for Efficient Gene Transfer and Expression in Manmmalian Cells, 3 Gene Ther. 448-457 (1996). However, despite their high transfection efficiency in vitro, inserting genes into the host cell's genome in this method depends on the viral infection pathway. The application of the viral infection pathway in application for human gene therapy introduces serious concerns about endogenous virus recombination, oncogenic effects, and inflammatory or immunologic reactions. G Ross et al., Gene Therapy in the United States: A Five-Year Status Report. 7 Hum. Gene Ther., 1781-1790 (1996). Because of these concerns, the use of viral vectors for human gene therapy has been extremely limited.
On the other hand, non-viral gene delivery systems such as cationic liposomes or synthetic gene carriers, e.g.poly-L-lysine (PLL), are being widely sought as alternatives. M. A. Wolfert et al., Characterization of Vectors for Gene Therapy Formed by Self-Assembly of DNA with Synthetic Block Co-Polymers. 7 Hum. Gene. Ther., 2123-2133 (1996); AV Kabanov & VA Kabanov DNA Complexes with Polycations for the Delivery of Genetic Materials into Cells. 6 Bioconj. Chem., 7-20 (1995). There are several advantages to the use of non-viral based gene therapies including their relative safety and low cost of manufacture. The major limitation of plasmid-based approaches has been that both the efficiency of gene delivery to several important somatic targets (i.e., liver and lung) and in vivo gene expression levels are lower using non-viral approaches than those using viral vectors.
Receptor-mediated gene delivery has its advantages and limitations. J. C. Perales et al., Biochemical and Functional Characterization of DNA Complexes Capable of Targeting Genes go [sic] Hepatocytes via the Asialoglycoprotein Receptor. 272 J. Biol. Chem.,7398-7407 (1997). Its advantages for use in gene therapy are as follows. First, the gene delivery carrier can be designed and customized for a specific target receptor. Second, the DNA does not have to integrate into the host cell genome to be expressed. Third, the delivery system is theoretically not limited by the size of the transgene. Finally, the technique does not involve the use of potentially infectious agents. There are also disadvantages that must be overcome before this procedure can be routinely used for human gene therapy. For example, the transgene is not integrated into the host cell chromosomes, or its expression is transient. Therefore, it will most likely be necessary to subject patients to multiple injections of a gene of interest. The DNA-ligand complexes are difficult to prepare and, until recently, little was known about their structure-function relationship. Also, there is only a fragmentary understanding of the biological process involved in the transfer of the transgene into the cell and its subsequent expression. These and other features of this system for gene therapy have recently been reviewed in detail. J. C. Perales et al., An Evaluation of Receptor-Mediated Gene Transfer Using Synthetic DNA-Ligand Complexes. 226 Eur. J. Biochem., 255-266 (1994).
In the mid 1970's, it was shown that PLL makes condensates with DNA. U. K. Laemmli Characterization of DNA Condensates Induces by Poly(ethylene oxide) and Polylysine. 72 Proc. Nat'l. Acad. Sci. U.S.A., 4288-4292 (1975). Since then, PLL modified with various substances, has been used as a gene carrier. (M. S. Wadhwa et al., Targeted Gene Delivery with a Low Molecular Weight Glycopeptide Carrier. 6 Bioconj. Chem., 283-291 (1995); A. Maruyama et al., (1997) Nanoparticle DNA Carrier with Poly(L-lysine) Grafter Polysaccharide Copolymer and Poly(D,L-lactic acid). 8 Bioconj. Chem., 735-742; G. Y. Wu, and C. H. Wu, Evidence for Targeted Gene Delivery to Hep G2 Hepatoma Cells in vitro., 27 Biochemistry, 887-892 (1988); P. Midoux et al., Specific Gene Transfer Mediated by Lactosylated Poly-L-Lysine into Hepatoma Cells. 21 Nucleic Acids Res., 871-878 (1993); P. Erbacher et al., Glycosylated Polylysine/DNA Complexes: Gene Transfer Efficiency in Relation with the Size and the Sugar Substitution Level of Glycosylated Polylysines and with the Plasmid Size. 6 Bioconj. Chem., 401-410 (1995).
PLL itself can be used as a DNA condensate. U.K. Laemmli, Characterization of DNA Condensates Induces by Poly(ethylene oxide) and Polylysine. 72 Proc. Nat'l. Acad. Sci. U.S.A. 72, 4288-4292 (1975)). However, the use of PLL alone as a gene delivery carrier has several disadvantages. First, its transfection efficiency is very low, because PLL has no functional group except the amine group used in charge-neutralization. Also, due to the negative charges of the DNA phosphate backbone, an increase in the degree of charge neutralization of the DNA often results in extensive condensation and the separation of the DNA phase in the form of insoluble compact structures. J. C. Perales et al., Biochemical and Functional Characterization of DNA Complexes Capable of Targeting Genes go [sic] Hepatocytes via the Asialoglycoprotein Receptor. J. Biol. Chem. 272, 7398-7407 (1997); D. E. Olinset al., Model Nucleoprotein Complexes: Studies on the Interaction of Cationic Homopolypeptides with DNA. 24 J. Mol. Biol., 157-176 (1967); J. T. Shapiro et al., Deoxyribonucleic Acid-Polylysine Complexes. Structure and Nucleotide Specificity. 8 Biochemistry 3219-3232 (1969).
In view of the foregoing, it will be appreciated that providing a targeted composition of gene therapy and a method for using thereof would be of a significant advancement in the art.